A number of physiological conditions and states are associated with increased levels of CK-MB, myoglobin, myosin and troponin. Elevated levels generally are associated with myocardial infarction and other conditions which result in myocardial injury.
Principally because of the association of increased levels of these proteins with acute myocardial infarction, tests for acute myocardial infarction (AMI) have been or are being devised to determine the level of these proteins in bodily fluids. Thus, these proteins have become known as cardiac markers.
Acute myocardial infarction continues to be a major cause of illness and death, particularly in the United States. An estimated 1.5 million admissions to hospitals can be attributed to suspected myocardial infarction or related cardiac disease. Of these patients, only roughly 25% are actually suffering an AMI while another 30% are admitted with unstable angina, 20% have stable coronary artery disease (CA), and the remaining 25% have no CAD. Differentiation of those patients who require immediate care and hospitalization from those who are not in danger is of great value in providing effective medical care, reducing hospitalization costs, and effectively managing hospital facilities. Current studies indicate that early intervention is critical for optimum therapeutic measures.
These therapeutic measures have the potential to restore blood flow to the damaged myocardium, limit the size of the infarct, and thus preserve cardiac function. New therapeutic intervention mechanisms, specifically thrombolytic agents such as streptokinase and tissue plasminogen activator are available to restore coronary artery blood flow and reduce the incidence of morbidity. Most clinicians believe that intervention must take place as soon as possible and should be well within the first four hours after the onset of chest pain.
Thus, an ideal cardiac marker or combination of markers should be cardiac tissue specific, it should be diagnostic within four hours after the onset of AMI, it should remain somewhat elevated for at least seven days after AMI but it should detect reinfarction even during the first few days of the first AMI.
Diagnosis of AMI is now based on an abnormal electrocardiogram (ECG), clinical symptoms and history, and elevated cardiac enzyme levels. Currently, CKMB is often used as the "definitive" serum marker for AMI.
However, often the ECG and clinical presentation give inconclusive or conflicting predictions of cardiac trauma. CK-MB testing has some limitations in contributing to final diagnosis. Skeletal muscle damage and strenuous exercise can artificially elevate CK-MB levels and confuse the clinical picture. In addition, CK-MB does not become diagnostically elevated until 4-6 hours after AMI. In addition, CK-MB levels become non-diagnostic 48-72 hours after AMI.
CK-MB has been prepared in a control solution to monitor diagnostic measurements of this analyte, however CK-MB is an enzyme which has limited stability in human serum and common buffered aqueous solutions. U.S. Pat. No. 4,994,375 discloses a stable reconstituted aqueous based control. Currently there are immunoassay kits, such as the DADE.RTM. STATUS.RTM. CK-MB Fluorometric Enzyme Immunoassay Kit available on the market for the determination of CK-MB levels. Many of these kits include calibrators. Control solutions containing CK-MB, such as the DADE.RTM. CK-MB/Myoglobin Immunoassay Control, are also commercially available.
Myoglobin is a marker present in both skeletal and cardiac muscle. Myoglobin levels are elevated within 2 hours of AMI. The serum level peaks in 6-8 hours but returns to non-diagnostic levels after 24-36 hours. However, serum myoglobin levels are also increased after skeletal muscle injury. There are a few immunoassay kits, such as the DADE.RTM. STRATUS.RTM. Myoglobin Fluorometric Enzyme Immunoassay Kit for the determination of myoglobin levels, that are commercially available. Myoglobin containing control solutions have been prepared and are commercially available from such sources as DADE.RTM. CK-MB/Myoglobin Immunoassay Control.
Troponin is a protein complex having a molecular weight of about 85 kD that performs the regulatory function of the contractile mechanism of the muscle tissue. The amino acid sequences of subunits which comprise the troponin complex has been determined. See, for instance, Vallins W. J. et al., Molecular cloning of human cardiac troponin I using polymerase chain reaction, FEBS LETTERS :Vol. 270, number 1, 2 Sep. 1990. Troponin is composed of three subunits of similar molecular weight, which, in the presence of calcium, cooperate to control either the contraction or relaxation of the muscle. The three subunits are designated troponin T, C, and I. Both the T and I molecules contained in heart muscle have amino acid sequences which are cardiac specific. Thus, both troponin T and troponin I have potential for superior specificity in testing for damage of myocardial origin. Damage to cardiac tissue causes these contractile proteins to be released into circulation fairly rapidly after injury providing the potential for sensitivity as well. Troponin is diagnostic 4-6 hours after AMI and remains elevated for 4-14 days.
Proteins of the contractile apparatus such as troponin are part of an insoluble protein complex. Thus, when purified troponin is placed in serum it is difficult to solubilize. In addition, purified preparations of troponin tend to be very labile and apparent changes in its conformation and/or adhesion to container surfaces tend to complicate quantification of the molecule. Thus, it is very difficult to design an aqueous solution which stabilizes troponin. Currently commercially available calibrators and controls used for diagnostic assays for troponin have very limited stability in liquid form.
Thus a need exists for aqueous solutions useful for solubilizing and stabilizing troponin. Such solutions can function as a diagnostic control or calibrator matrix for troponin and other cardiac markers or other proteins that are difficult to solubilize and/or stabilize. In addition, the matrix is useful for storing the protein(s).
Several criteria need to be met when formulating a calibrator or control base for troponin or other proteins that have stability or solubility issues similar to troponin. Stability issues are of primary concern. Liquid products are preferred for reproducibility and ease of use and should be stable. However if the product is lyophilized, it should be stable after reconstitution. Previous troponin calibrators are based on human serum derived products and contribute very little to the stability of the composition. For instance, the published "dating" of human serum based lyophilized troponin T calibrators and controls of the Boehringer Mannheim ELISA-TEST.RTM. Troponin T after reconstitution is only 6 hours at 2 to 8 C and 3 months when aliquoted and stored at -20 C. A matrix which increases the stability of the product is highly desirable.
Moreover, use of normal or processed human serum presents health issues to both clinicians and manufacturers. Thus, a matrix which lowers health risks is also highly desirable.
The calibrators in a synthetic matrix must mimic the shape of a response curve using normal human serum. This is important to ensure that results read off a standard curve generated with the matrix are accurate when comparing the results to the actual biological milieu.
In a diagnostic assay, non-specific binding of the analyte to the test surface (e.g. solid support such as test tubes, paper, slides etc.) must be minimized in order to keep calibration accurate and eliminate any risks of "discrepant" results. Thus, the non-specific binding of the analyte in a matrix must be minimized and must be similar to the non-specific binding of samples. It is also important that during storage, the protein or protein fragment does not appreciably bind to the storage container.
There are instances when an analyte analogue may be more desirable than the actual analyte. If an analyte analogue is used instead of the analyte, the binding of the analogue must mimic the binding of the analyte. Particular care must be used when selecting analogues for proteins because the immunobinding of the analogue must mimic that of the protein. Thus, any conformational dependence of the protein for the binding site must be maintained in the analogue. In addition, stability of the analogue should be the same or greater than that of the analyte. Again, the stability of the protein analyte may be related to its conformation. Finally, if an analogue is substituted for an analyte, it is desirable that the analogue be more readily available than the analyte.